RESUMEN
Genetic and biochemical evidence demonstrated that Eps8 is involved in the routing of signals from Ras to Rac. This is achieved through the formation of a tricomplex consisting of Eps8-E3b1-Sos-1, which is endowed with Rac guanine nucleotide exchange activity. The catalytic subunit of this complex is represented by Sos-1, a bifunctional molecule capable of catalyzing guanine nucleotide exchange on Ras and Rac. The mechanism by which Sos-1 activity is specifically directed toward Rac remains to be established. Here, by performing a structure-function analysis we show that the Eps8 output function resides in an effector region located within its COOH terminus. This effector region, when separated from the holoprotein, activates Rac and acts as a potent inducer of actin polymerization. In addition, it binds to Sos-1 and is able to induce Rac-specific, Sos-1-dependent guanine nucleotide exchange activity. Finally, the Eps8 effector region mediates a direct interaction of Eps8 with F-actin, dictating Eps8 cellular localization. We propose a model whereby the engagement of Eps8 in a tricomplex with E3b1 and Sos-1 facilitates the interaction of Eps8 with Sos-1 and the consequent activation of an Sos-1 Rac-specific catalytic ability. In this complex, determinants of Eps8 are responsible for the proper localization of the Rac-activating machine to sites of actin remodeling.
Asunto(s)
Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Proteínas/metabolismo , Proteína SOS1/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteínas ras/metabolismo , Animales , Proteínas Portadoras/genética , Fraccionamiento Celular , Extensiones de la Superficie Celular , Células Cultivadas , Medio de Cultivo Libre de Suero , Citocalasina D/farmacología , Proteínas del Citoesqueleto , Embrión de Mamíferos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Genes Reporteros , Humanos , Immunoblotting , Péptidos y Proteínas de Señalización Intracelular , Ratones , Microscopía Fluorescente , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Estructura Terciaria de Proteína , Proteínas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína SOS1/genética , Transducción de Señal/fisiología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Transfección , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rac/genética , Proteínas ras/genéticaRESUMEN
Members of a family of intracellular molecular switches, the small GTPases, sense modifications of the extracellular environment and transduce them into a variety of homeostatic signals. Among small GTPases, Ras and the Rho family of proteins hierarchically and/or coordinately regulate signaling pathways leading to phenotypes as important as proliferation, differentiation and apoptosis. Ras and Rho-GTPases are organized in a complex network of functional interactions, whose molecular mechanisms are being elucidated. Starting from the simple concept of linear cascades of events (GTPase-->activator--> GTPase), the work of several laboratories is uncovering an increasingly complex scenario in which upstream regulators of GTPases also function as downstream effectors and influence the precise biological outcome. Furthermore, small GTPases assemble into macromolecular machineries that include upstream activators, downstream effectors, regulators and perhaps even final biochemical targets. We are starting to understand how these macromolecular complexes work and how they are regulated and targeted to their proper subcellular localization. Ultimately, the acquisition of a cogent picture of the various levels of integration and regulation in small GTPase-mediated signaling should define the physiology of early signal transduction events and the pathological implication of its subversion.
Asunto(s)
GTP Fosfohidrolasas/fisiología , Transducción de Señal/fisiología , Animales , GTP Fosfohidrolasas/clasificación , Guanosina Trifosfato/fisiología , Mamíferos/metabolismo , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas de Unión al GTP rac/fisiología , Proteínas ras/fisiología , Proteínas de Unión al GTP rho/fisiologíaRESUMEN
The small guanine nucleotide (GTP)-binding protein Rac regulates mitogen-induced cytoskeletal changes and c-Jun amino-terminal kinase (JNK), and its activity is required for Ras-mediated cell transformation. Epistatic analysis placed Rac as a key downstream target in Ras signalling; however, the biochemical mechanism regulating the cross-talk among these small GTP-binding proteins remains to be elucidated. Eps8 (relative molecular mass 97,000) is a substrate of receptors with tyrosine kinase activity which binds, through its SH3 domain, to a protein designated E3b1/Abi-1. Here we show that Eps8 and E3b1/Abi-1 participate in the transduction of signals from Ras to Rac, by regulating Rac-specific guanine nucleotide exchange factor (GEF) activities. We also show that Eps8, E3b1 and Sos-1 form a tri-complex in vivo that exhibits Rac-specific GEF activity in vitro. We propose a model in which Eps8 mediates the transfer of signals between Ras and Rac, by forming a complex with E3b1 and Sos-1.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas/metabolismo , Transducción de Señal , Proteínas ras/metabolismo , Células 3T3 , Animales , Células COS , Clonación Molecular , Proteínas del Citoesqueleto , GTP Fosfohidrolasas/metabolismo , Vectores Genéticos , Factores de Intercambio de Guanina Nucleótido , Guanosina Trifosfato/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Ratones , Unión Proteica , Proteínas/genética , Proteínas de Unión al GTP rac , Factores de Intercambio de Guanina Nucleótido rasRESUMEN
To gain insight into the regulatory mechanisms and the signals responsible for the adaptation of higher plants to conditions of varying sulfate availability, we have isolated from a sulfate-deprived root library maize cDNAs encoding sulfate permease (ZmST1) and ATP sulfurylase (ZmAS1), the two earliest components of the sulfur assimilation pathway. The levels of ZmST1 and ZmAS1 transcripts concomitantly increased in both roots and shoots of seedlings grown under sulfate-deprived conditions, and rapidly decreased when the external sulfate supply was restored. This coordinate response, which was not observed under conditions of limiting nitrate or phosphate, correlated with the depletion of glutathione, rather than sulfate stores. However, drastically reducing glutathione levels through treatment with buthionine sulfoximine, a specific inhibitor of gamma-glutamyl cysteine synthetase, did not provide an adequate stimulus for the up-regulation of either sulfate permease or ATP sulfurylase messengers. Indeed, L-cysteine, but not D-cysteine, effectively down-regulated both transcripts when supplied to sulfur-deficient seedlings under conditions of blocked glutathione synthesis. Altogether, these data provide evidence for the coordinate regulation of sulfur assimilation mRNAs in higher plants and for the glutathione-independent involvement of cysteine as a stereospecific pretranslational modulator of the expression of sulfur status-responsive genes.
Asunto(s)
Proteínas de Transporte de Anión , Proteínas de Transporte de Membrana/genética , ARN Mensajero/genética , Sulfato Adenililtransferasa/genética , Azufre/metabolismo , Zea mays/genética , Cisteína/química , Cisteína/farmacología , ADN Complementario/química , ADN Complementario/genética , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Glutatión/metabolismo , Datos de Secuencia Molecular , Plantas/efectos de los fármacos , Plantas/enzimología , Plantas/genética , Análisis de Secuencia de ADN , Estereoisomerismo , Sulfatos/metabolismo , Zea mays/química , Zea mays/enzimologíaRESUMEN
A family with a very high prevalence of pheochromocytoma (62%, i.e. 100% in the 1st generation and 75% of the second one) is described. The proband was a 19-year-old woman with a 30 g right-side tumor, who died at 24 years because of a heart failure. Both sisters of the proband developed a right-side pheochromocytoma at 13 and respectively 14 years of age and are now normotensive after surgical exeresis. One of them had 3 sons: 2 with pheochromocytoma and a third one dead at 9 years of age for pulmonary oedema. Accuracy is needed in studying relation of all subjects with pheochromocytoma.